Influenza vaccine with reduced pyrogenicity

ABSTRACT

An inactivated influenza virus vaccine is produced by treating the virus before or after inactivation with formalin with a small amount of an organic solvent selected from the group consisting of dilower alkyl ethers and lower alkyl esters of lower fatty acids. The amount was usually less than 2 volumes of the solvent, for a short time, not significantly in excess of 4 hours and preferably from 15 minutes to 2 hours, the shorter times being used when the volume of solvent is in excess of 1. Preferred solvents are diethyl ether and methyl acetate. In the case of the smaller amounts of solvent, such as for example one-eighth the volume of the aqueous virus concentrate, it is not necessary to decant the solvent phase; it may be removed by blowing nitrogen through the mixture. With larger amounts of solvent decantation is desirable, followed by removing the last traces with nitrogen bubbled through the liquid. If the solvent treatment is prior to inactivation with formalin, inactivation then follows, and the vaccine is preserved and stored in the usual manner, for example with a small amount of merthiolate. The vaccine has a titer not significantly less than untreated virus whether measured by either the chick cell agglutination (CCA) test, or the hemagglutinin (HA) test. The pyrogenicity is, however, reduced to a very low point. Rabbit tests show temperature rises of less than 0.4* C. as against temperature rises of 1* C. or somewhat more in untreated vaccines.

United States Patent [72] Inventor Arden WesleyMoyer 138 Fremont Ave., Park Ridge, NJ. 07645 [2|] Appl. No. 644,812 [22] Filed June 9, 1967 [45] Patented Dec. 14, 1971 [54] INFLUENZA VACCINE WITH REDUCED Primary Examiner-Richard L. Huff Attorney-Norton S. Johnson ABSTRACT: An inactivated influenza virus vaccine is produced by treating the virus before or after inactivation with formalin with a small amount of an organic solvent selected from the group consisting of dilower alkyl ethers and lower alkyl esters of lower fatty acids. The amount was usually less than 2 volumes of the solvent, for a short time, not significantly in excess of4 hours and preferably from 15 minutes to 2 hours, the shorter times being used when the volume of solvent is in excess of l. Preferred solvents are diethyl ether and methyl acetate.

In the case of the smaller amounts of solvent, such as for example one-eighth the volume of the aqueous virus concentrate, it is not necessary to decant the solvent phase; it may be removed by blowing nitrogen through the mixture. With larger amounts of solvent decantation is desirable, followed by removing the last traces with nitrogen bubbled through the liquid. If the solvent treatment is prior to inactivation with formalin, inactivation then follows, and the vaccine is preserved and stored in the usual manner, for example with'a small amount of merthiolate. The vaccine has a titer not significantly less than untreated virus whether measured by either the chick cell agglutination (CCA) test, or the hemagglutinin (HA) test. The pyrogenicity is, however, reduced to a very low point. Rabbit tests show temperature rises of less than O.4C. as against temperature rises of 1C. or somewhat more in untreated vaccines.

BACKGROUND OF THE INVENTION Fever produced from influenza virus vaccines is fairly common among children and only somewhat less so among adults. lt has been proposed in the past to treat the vaccine with diethyl ether, for example 2 volumes of diethyl ether per volume of vaccine concentrate, at low temperatures for a long period of time, for example hours and more. After separating the ether phase and removing residual ether by blowing nitrogen through to remove the last traces of the ether, the virus is inactivated with formalin, (for example, a concentration of 1:1000), a small amount of a preservative, merthiolate, added, and then afier centrifuging and dialyzing, a dilution in standard isotonic saline is made. The pyrogenicity is somewhat reduced and a reasonable virus titer remains, though often not as high as untreated virus. Somewhat shorter treatment times with the ether in the same higher concentrations have also been proposed.

In the diethyl ether treatment of the prior art it was considered necessary to carry out this treatment on the uninactivated virus and to inactivate it only after removing the diethyl ether. For some purposes this is inconvenient, an it would be desirable to inactivate the virus first and then remove pyrogens.

SUMMARY or THE INVENTION According to the present invention, influenza virus, before or after inactivation with fonnalin, is treated with much lower volume of a solvent selected from the group consisting of dilower alkyl ethers and lower alkyl esters of lower fatty acids. The treatment is also for a much shorter time. Optimum time varys with the amount of solvent used, being very short, for example, 15 minutes with amounts of solvent approaching those which were hitherto used. Somewhat longer times, up to several hours, are pennissible with much smaller amounts of solvent, for example one-eighth the volume of the virus concentrates. In each case satisfactory reduction of pyrogenicity is effected, and animal protency remains high, up to five times the potency obtainable with the untreated vaccine. Improved, low pyrogenic vaccines of high potencies are obtained, regardless of whether the treatment with the solvent is before or after inactivation of the virus with formalin. In the process of the present invention no difference is noted whether the treatment is carried out with live virus or inactivated virus.

While there are dilower alkyl ethers, such as dimethyl, diethyl or dipropyl ethers, and esters, such as methylor ethylacetate, which are useful, itis preferred to use diethyl ether or methyl acetate; and in a more specific aspect of the invention these preferred procedures are included.

it is an advantage of the present invention that it is not necessary to filter active influenza virus concentrate, which was formerly considered necessary, using a column with a cross-linked dextran in bead form, usually known under its trade name of Sephadex. Such preliminary treatment is not excluded from the present invention, but it is an advantage that satisfactory products can be obtained without this additional step.

lt is desirable, although not essential, that a small amount of a wetting agent be added, for example 1 mg./ml. of sorbitan monooleate. The amount of wetting agent is not at all critical, and where it is considered that sorbitan monooleate might conceivably have some undesirable side effects, corresponding amounts of other wetting agents which are physiologically acceptable may be used. A wetting agent is preferred, but this is not the distinction from the prior processes, which also used wetting agents. in common with prior proposals, it is desirable, although again not essential, that fairly low temperatures be used in the treatment, for example about 4 C. The particular temperature is not the distinction of the present invention over the prior art but is mentioned here merely as indicating good practice, which should be followed in the process of the present invention just as in other treatments.

The making up of a final vaccine after the solvent treatment does not differ from that of the prior art. lt normally involves the use of small amounts of preservative, such as merthiolate, dialysis, and making up to standard concentration in isotonic saline. The present invention can be considered as stopping when the high potency, low pyrogen product is produced. it is an advantage that no new steps or procedures in the final processing or packaging are needed and more or less conventional procedures may be employed.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Typical preferred embodiments will be described in conjunction with the following specific examples, in which the standard abbreviations CCA for Chick Cell Agglutination and HA for Hemagglutin ae used. Also, where pyrogenicity tests are employed, the standard criterion of a temperature rise of not over 0.4 C., in rabbitsv is considered the borderline between satisfactory and unsatisfactory removal of pyrogens.

EXAMPLE 1 Virus concentrate from the PR 8 strain obtained from Ann Arbor, Mich., was made up with 0.1 percent sorbitan monooleate. The influenza was of C. A, and the final concentration of wetting agent was 1 mg./m1. of vaccine.

A 0.5 volume of diethyl ether was added and the mixture stirred constantly for 1 hour at 4-5 The temperature is not critical but should be less than 37 C. The mixture was then allowed to stand overnight and separated into two phases. The ether phase was decanted and discarded, and the aqueous phase was then aerated with nitrogen for 1 hour to remove residual ether. It was tested by the HA and CCA tests and by the standard pyrogen test, done intravenously in rabbits. The results appear in the following table:

'1 B E 1.--'IREATMENT OF LIVE INFLUENZA VIRUS A L CONCENTRATE WITH DIE'IHYL ETHER it will be apparent that the virus titer was at least as great and in the case of CCA values about 50 percent greater than the untreated control. Pyrogenicity was satisfactory whereas the control was unsatisfactory.

The potency of the vaccine were determined by the following tests:

The vaccines both untreated and ether treated were diluted, serially varying by a factor of S, i.e., 1:5 1:25 1:125 1:625 and 1:3125. Each dilution of each vaccine was administered to groups of five mice. After 2 weeks the mice were bled and the serum from each group was administered with 158 LD, as a challenge dose to a second group of mice. 1f there were sufficient neutralizing antibodies in the serum challenge in each case the mice should survive. At a dilution of 1:625 in the case of the untreated vaccine, none of a group of six mice survived, at a dilution of 1:125, six out of six mice survived. However, at a dilution of 1:625 of the ether treated vaccine five out of five mice survived. In other words the ether treated vaccine could be diluted five times further than the untreated vaccine and still stimulate sufficient antibodies to protect against the challenge dose. Such tests are often referred to as potency tests.

EXAMPLE II The procedure of example 1 was repeated except that a type B influenza Lee strain was used. Both live and formalin-inactivated preparations were used. A 0.125 volume of methyl acetate was added and the mixture stirred constantly for about 1 hour at room temperature. The methyl acetate was then removed by aeration with nitrogen gas for approximately 1.5

hours and potency and pyrogenicity tested as in example I. The results are as follows:

TABLE 4,-EFFECT OF TIME OF DIETI-IYL ETIIER TREAT- MENT ON POTENGY AND PYROGENIC PROPERTIES OF INFLUENZA VIRUS CONCENTRATES Concen- Time of tration of treat- Virus titer Pyroge- TABLE 2.TREATMENT OF INFLUENZA VIRUS CONOEN- 5 Influenza diethyl ment, nicity, WITH METHYL ACETATE PLUS SORBITAN MONO- type ether hour GOA/ml. IIA/ml. C. O E

Mary1and Nonecon- 3, 888 6,400 l. 3 Concen- Time and trol, tration of temper- Virus titer Pyro- D 2 volumes 0. 5, 054 0, 400 0. 3 methyl ature of gonicity, 'Do d0 1.0 4, 025 6,400 0. 1 Tnfluenza tyne acetate treatment HA CCA C. Do do 1.5 0 400 0. 11 L00 (live) 0.125 1 hr., 23 12, 800 10,000 1.4 Avorngn of 3 rabbits.

C r v new. Do Control untrenterl 12,500 0, 400 1.10 L00 (l|mcLlv:il.t-xl).... 0.125 4 hrs., 22 12,800 10,000 .07 It be that very of treatment hour (1. the potency for HA of the vaccine was as good as or better 00mm than the untreated control. At l hour the figures were substan- 1 Average of 3 rabbits.

It will be noted that regardless of whether the vaccine was inactivated prior to treatment with the methyl acetate or was not inactivated, the virus titer remained as high for HA as the untreated controls in the case of the uninactivated control, and there was an actual increase of CCA even when the inactivated virus was treated for the extreme period of 4 hours, the virus titer remained about the same. Pyrogenicity was unsatisfactory with the uninactivated control, borderline with the inactivated control, and very low and, therefore, satisfactory with the vaccines which had received the methyl acetate treatment.

EXAMPLE Ill The general procedure of example I was repeated with a Maryland strain of virus, concentrated 20 times, using various concentrations of diethyl ether.

Average of 3 rabbits. up h It will seem that with 2 volumes of diethyl ether, while low.

pyrogenicity was obtained, the potency was considerably less than the untreated control. A marked improvement, about double the potency, resulted when 1 volume of diethyl ether was used, and when 0.5 volume was used, the potency for HA was as great as the untreated control, and actually markedly increased for CCA.

EXAMPLE IV The efiect of time of diethyl ether treatment was tested with relatively large volumes, 2 volumes, using the procedure of example ill. The results appear in the following table:

tially the same, but at 1.5 hours the potency had dropped off to zero for CCA and to an extremely low and useless figure for HA. As might'be expected, pyrogenicity was good in the case of all of the treated material.

EXAMPLE V A mixed vaccine concentrate with Maryland, Ann Arbor, Taiwan, PR 8 and Japanese 170, was treated with diethyl ether. Virus titers were essentially the same after treatment, but the pyrogenicity was lowered. The results appear in the following table:

Table 5.TREATMENT OF INFLUENZA VIRUS CONCEN- g t tg n wrrrr DIETHYL-ETHER PLUS SORBITAN MONO- :Ax eewwe The potency of the diethyl ether treated mixed vaccines was tested against standard NlH vaccine. As in the case of example i, the vaccines were serially diluted to the same dilutions, injected into mice and two weeks later the mice were bled and the blood centrifuged to collect the serum. The serum from each group was then combined with a challenge dose of one of the five strains of influenza virus and inoculated into a new group of mice. As in the case of example I, survival of the mice shows that the vaccine produced adequate antibodies. At a dilution of 1:125 serum from the mice vaccinated with the diluted diethyl ether treated vaccine protected six out of six mice from the challenged dose of the PR 8. At the same dilution serum from mice vaccinated with the control vaccine, NIH failed to protect any of the six mice.

I claim: 7

1. A process for preparing an improved influenza vaccine of low pyrogenicity and high potency, which comprises the steps of: treating the virus concentration at a temperature of from 4 C., to room temperature with from 0.05 volume to less than 1 volume of methyl acetate for a period of from 15 minutes to 2 hours; separating the aqueous phase; and preparing a vaccine from said aqueous phase.

2. A process according to claim 1, in which the treatment with methyl acetate is of live virus and the virus is inactivated with fonnalin prior to vaccine formation.

3. A process according to claim 1, in which the virus is inactivated prior to treatment with methyl acetate. 

2. A process according to claim 1, in which the treatment with methyl acetate is of live virus and the virus is inactivated with formalin prior to vaccine formation.
 3. A process according to claim 1, in which the virus is inactivated prior to treatment with methyl acetate. 